Design of new medium for the production of Mannheimia haemolytica leukotoxin and its characterization

Leukotoxin is considered the most important virulence factor in the pathogenesis of pneumonic pasteurellosis which is caused by M. haemolytica. This exotoxin has a toxic activity against ruminant leukocytes and also plays a central role in the disease progression. The immune response developed in the animal's body against leukotoxin is effective in protecting against the disease. So, this study was aimed to develop media for M. haemolytica A1 (ATCC BAA 410) leukotoxin and antigenic characterization of the obtained leukotoxin. Statistical design was used to evaluate the effect of 24 components in leukotoxin production which are 15 amino acids, 3 vitamins, 3 inorganic salts and 2 other components. Two-stage of Plackett-Burman Design (PBD) using Design Expert program was conducted to determine the components with low, positive or negative contribution on leukotoxin production which are L-proline, L-threonine, L-valine, L-glutamine and NH4SO4. Components with low contribution on leukotoxin production were eliminated and second stage of PBD was carried out to identify the most important components on leukotoxin production. In the second stage of PBD, L-arginine, L-isoleucine, L-glutamic acid and phenylalanine were identified as components with highly positive or negative effect in leukotoxin production. The optimal concentrations of these components were determined using CCD as fallowing: L-arginine, 0.69 g/L; L-isoleucine, 1.4 g/L; L-glutamic acid, 3.59 g/L and L-phenylalanine, 0.058 g/L. The validation experiments were carried out also to confirm the CCD model. Bacterial growth was measured spectrophotometrically and the amount of leukotoxin was measured by ELISA using monoclonal antibody. It was observed that when K2HPO4, NaHCO3 and NaHPO4 were used to further improve the designed media, cell growth and leukotoxin production were increased. The best biomass production of M. haemolytica growth was observed in BHI when compared to the designed media and RPMI-1640. However, when evaluated for leukotoxin production it was observed that leukotoxin production was less in BHI (16μg/ml) than designed media (18μg/ml) and RPMI-1640 medium (43μg/ml). Additionally, it is the first report on the effect of L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine and tiamin HCl in leukotoxin production. Keywords: leukotoxin, Mannheimia haemolytica, statistical optimization, Plackett-Burman design, Central Composite Design.